Essential Oils Derived from Cistus Species Activate Mitochondria by Inducing SIRT1 Expression in Human Keratinocytes, Leading to Senescence Inhibition

Cistus L. is a genus of dicotyledonous perennial herbaceous plants. Cistus species have been commonly used in folk medicine in the Mediterranean region. In the present study, the biological activities of essential oils derived from Cistus species (Cistus laurifolius, C. monspeliensis, C. creticus, and C. salviifolius) were evaluated. Essential oils derived from C. laurifolius and C. monspeliensis were found to augment the expression of SIRT1, an anti-aging gene, in the normal culture of HaCaT cells. Furthermore, these essential oils increased the number and size of mitochondria and augmented their activity. These effects were thought to be caused by the up- and downregulated expression of MITOL and Drp1 in HaCaT cells, respectively, in response to the essential oil treatment. In addition, these essential oils were found to attenuate ultraviolet-B-induced mitochondrial damage and cellular senescence in HaCaT cells. These findings indicate that essential oils derived from C. laurifolius and C. monspeliensis may inhibit skin aging through mitochondrial regulation via SIRT1 activation.


Introduction
Cistus L., commonly known as rock rose, is a genus of dicotyledonous perennial herbaceous plants that have hard leaves, grow in open areas with stony and infertile soils, and are indigenous to the Mediterranean region [1]. The family of Cistaceae consists of 8 genera and 180 species. Cistus species have been commonly used in folk medicine as herbal tea, extracts, and fragrances in the Mediterranean region, and specifically in Morocco [2]. Recently, phytochemical profiles and the various pharmacological activities of Cistus species were reviewed, where the antimicrobial activities, including antiviral, antiparasitic, antifungal, and antibacterial potentials, of essential oils, raw extracts, and isolated compounds were introduced [3]. In the present study, four Cistus species from the High Atlas Mountains of Morocco (Cistus laurifolius, C. monspeliensis, C. creticus, and C. salviifolius) which are known to be rich in phenylpropanoids and terpenes were used. Several flavonoids, including quercetin, myricetin, kaempferol, and apigenin and their aglycones, tannins, and ellagitannins, were detected in the leaves [4]. These plants contain many biologically active compounds that are expected to have a variety of functional properties, such as antioxidant activity. In the present study, the anti-aging activities of essential oils derived from the four Cistus species on skin keratinocytes were evaluated. SIRT1 is a mammalian ortholog of a yeast silent information regulator 2 (Sir2). It is an NAD + -dependent deacetylase that mediates the effects of calorie restriction and regulates the lifespan of several organisms. SIRT1 has recently received much attention as an anti-aging gene [5]. In particular, it has been reported that SIRT1 can achieve anti-aging at the cellular level by enhancing mitochondrial biosynthesis through the activation of PGC-1α [6].
Previously, novel systems for screening food and food ingredients with anti-aging activities by targeting SIRT1 were developed. In those reports, polyphenols derived from the pomegranate were found to repair ultraviolet-B-induced DNA damage via SIRT1 activation [7,8]. Using these systems, the novel functions of essential oils derived from Cistus species were evaluated.

Identification of Essential Oil That Activates SIRT1 Transcription
Among these five essential oils, essential oils derived from leaves of C. laurifolius before flowering, C. salviifolius, and C. monspeliensis strongly activated the SIRT1 promoter in Ha-CaT cells ( Figure 1A), although essential oils derived from the leaves of C. laurifolius during flowering and C. creticus did not. Next, the effects of the essential oils from C. laurifolius before flowering and C. monspeliensis on the expression of endogenous SIRT1 in HaCaT cells ( Figure 1B,C) were evaluated. The results showed that essential oils significantly augmented the expression of endogenous SIRT1 in HaCaT cells both at the mRNA level and at the protein level.  100 ng/mL) were added to the HaCaT (SIRT1p-EGFP) cells and cultured for 48 h; then, changes in EGFP fluorescence were monitored. (B) Effects of essential oils on the expression of endogenous SIRT1 in HaCaT cells evaluated by qRT-PCR. Statistical significance was determined by using a two-sided Student's t-test. Statistical significance was evaluated by comparison with the control and defined as p < 0.05 (* p < 0.05; *** p < 0.001). Values are means ± SEM (n = 3). (C) Effects of essential oils on the expression of SIRT1 protein in HaCaT cells evaluated by Western blot. As a control treatment, HaCaT cells were treated with dimethyl sulfoxide (DMSO).

Effects of Cistus Essential Oils on the Mitochondrial Biogenesis and Differentiation
Peroxisome-proliferator-activated receptor gamma coactivator 1-α (PGC-1α) functions as a downstream factor of SIRT1 and is known to be a master regulator of mitochondrial biogenesis. As shown in Figure 2A, the essential oils increased the endogenous expression of PGC-1α in HaCaT cells. Then, the effect of essential oil on mitochondrial biogenesis was evaluated. Consistently, the essential oils increased the number and/or size of mitochondria, as evidenced by the increased copy number of mitochondrial DNA in HaCaT cells treated with these essential oils ( Figure 2B). Furthermore, these essential oils augmented the expression of genes (involucrin, loricrin) relating to skin cell differentiation ( Figure 2C,D), suggesting that these essential oils can enhance the function of the cell envelope and barrier.  Statistical significance was determined by using a two-sided Student's t-test. Statistical significance was evaluated by comparison with the control and defined as p < 0.05 (* p < 0.05; *** p < 0.001). Values are means ± SEM (n = 3). As a control treatment, HaCaT cells were treated with DMSO. Furthermore, in order to clarify the effects of essential oil on mitochondrial biogenesis and activity, fluorescent probes of MitoTracker Red CMXRos and MitoTracker Green FM were used. MitoTracker Red CMXRos fluorescence was dependent on mitochondrial membrane potential, whereas MitoTracker Green FM was not affected by mitochondrial membrane potential [9]. Thus, mitochondrial activity can be estimated by MitoTracker Red CMXRos fluorescence, and mitochondrial count and size can be measured using Mito-Tracker Green FM fluorescence by using IN Cell Analyzer 2200. An example of MitoTracker staining is shown in Figure 3A. Our results showed that these essential oils significantly activated mitochondria and increased their number and size in HaCaT cells ( Figure 3B), suggesting that the essential oils regulate the morphological stability of mitochondria. . Statistical significance was determined by using a two-sided Student's t-test. Statistical significance was evaluated by comparison with the control of each experiment and defined as p < 0.05 (* p < 0.05; *** p < 0.001). Values are means ± SEM (n = 3). As a control treatment, HaCaT cells were treated with DMSO.
Next, the effects of these essential oils on the morphological stability of mitochondria were evaluated by investigating the expression of MITOL, a mitochondrial ubiquitin ligase, and Drp1, a mitochondrial fission factor [10]. The essential oils augmented the expression of MITOL and decreased the expression of Drp1, suggesting that they stabilize mitochondrial morphology ( Figure 4A,B).

Effects of Cistus Essential Oils on UVB-Induced Mitochondrial Damage and Senescence
As shown in Figure 5, UVB irradiation significantly induced mitochondrial damage. UVB reduced mitochondrial activity and reduced the number and size of mitochondria in HaCaT cells. However, essential oils greatly attenuated UVB-induced mitochondrial damage in HaCaT cells. Statistical significance was determined by using a two-sided Student's t-test. Statistical significance was evaluated by comparison with the control irradiated with UVB and defined as p < 0.05 (* p < 0.05; *** p < 0.001). Values are means ± SEM (n = 3).
As shown in Figure 6, UVB treatment significantly induced cellular senescence in Ha-CaT cells. However, essential oils significantly attenuated UVB-induced cellular senescence in HaCaT cells. Figure 6. Effects of essential oils on UVB-induced cellular senescence. HaCaT cells were irradiated with 10 mJ/cm 2 and then treated with essential oils (final concentration: 100 ng/mL) for 48 h. The effects of essential oils on the UVB-induced cellular senescence were evaluated by using a fluorescent substrate of β-galactosidase, C12FDG. Statistical significance was determined by using a two-sided Student's t-test. Statistical significance was evaluated by comparison with the control irradiated with UVB and defined as p < 0.05 (* p < 0.05; ** p < 0.01; *** p < 0.001). Values are means ± SEM (n = 3).

Discussion
To date, the leaf extracts and flowers of Cistus species have many functions in preventing various diseases and maintaining good physical condition [11][12][13][14], which may be the reasons why they are used in traditional folk medicine to improve inflammatory conditions. In the present study, essential oils derived from Cistus species were shown to increase mitochondrial biogenesis and activity and suppress cellular senescence induced by UVB through the activation of SIRT1. Previous studies have shown that activation of SIRT1 in skin cells repairs UV-induced DNA damage [7] and enhances the synthesis of ceramide [15,16], which is responsible for the internal skin barrier. Furthermore, it has been shown that mitochondrial activation associated with SIRT1 activation in skin cells contributes to the suppression of wrinkles [10]. Based on the above studies, the essential oil used in this study is thought to have skin improvement effects, such as skin barrier enhancement, wrinkle suppression, and repair of UVB-induced damage, through the activation of SIRT1.
Compositional analyses of these essential oils have been conducted by various researchers [17,18]. Recently, the chemical composition of the essential oil of Cistus species has been investigated using GC-MS and other techniques. Many sesquiterpenes and monoterpenoids have been identified, and it is thought that these components may explain the various functions including antimicrobial, anti-inflammatory, and antioxidant properties of the essential oil, but direct evidence is still scarce [1,6,19]. However, the effects of Cistus essential oil on skin cells, especially on skin SIRT1 and mitochondria, have not yet been studied; thus, the present study is a novel report that reveals new functions of Cistus essential oil on the skin. In addition, analyses to identify the components of the essential oils of C. laurifolius and C. monspeliensis involved in skin SIRT1 and mitochondrial activation are in progress, and new components that function in skin cell activation can be found in the near future.
This study revealed that the screening system used in this study is useful in the search for foods and food ingredients with anti-aging activity. Using this system, the antiaging activities of plant extracts as well as polyphenols and lactic acid bacteria have been discovered [7,8,20]. In the present report, we used the promoter activity of an anti-aging gene as an indicator and skin cells as evaluation cells to evaluate the functionality of Cistus essential oil. However, if other anti-aging genes such as SIRT3 and telomerase are used as target genes and/or different cells (intestinal cells, muscle cells, brain cells) are used for screening, it will be possible to find new functionalities of Cistus essential oil [21].

Plant Materials and Essential Oil Extraction
The leaves of C. laurifolius specimens were collected before and during plant flowering The leaves of C. laurifolius, C. monspeliensis, C. creticus, and C. salviifolius were shadedried and coarsely ground, and aliquots of powder (100 g) of each plant were individually used to obtain the essential oils by steam distillation for 4 h. The essential oils were recovered from the hydrolysate by liquid-liquid extraction using n-hexane. The solvent was removed under reduced pressure using a rotary evaporator at 40 • C. The essential oils were dried over anhydrous sodium sulfate and stored at 4 • C in dark-colored stoppered glasses until use. The yield of essential oil is the ratio of the weight of the extracted oil obtained after evaporation and that of the plant material used. The yields obtained from C. laurifolius, C. monspeliensis, C. creticus, and C. salviifolius were 0.127%, 0.1%, 0.078%, and 0.08%, respectively.

Cell Line and Treatment
The HaCaT human keratinocyte cell line (Riken Bioresource Center, Tsukuba, Japan) was cultured in Dulbecco's modified Eagle's medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Life Technologies, Gaithersburg, MD, USA) at 37 • C in a 5% CO 2 atmosphere. Resveratrol and essential oils were dissolved in 100% DMSO at the concentrations of 10 mM and 100 µg/mL, respectively. These stock solutions were added to HaCaT cells to make a 1000-fold dilution. Therefore, in control experiments, 100% DMSO was added to HaCaT cells to make a 1000-fold dilution. Cells were treated with essential oils at the final concentration of 100 ng/mL by 1000-fold dilution of stock solution with culture medium. Resveratrol was used as a positive control to enhance SIRT1 expression in HaCaT cells.

DNA Isolation
HaCaT cells (6.0 × 10 4 cells/mL) were seeded onto a culture dish (60 mm × 15 mm) and cultured for 24 h. The cells were further cultured for 72 h with daily addition of 10 µM resveratrol or 100 ng/mL essential oil. DNA was extracted from HaCaT cells using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. DNA content was measured using a NanoDrop 2000/2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Determination of Mitochondrial DNA (mtDNA) Copy Number
qRT-PCR analyses were performed to determine the mtDNA copy number in samples using the Human Mitochondrial DNA (mtDNA) Monitoring Primer Set (Takara) in duplicates. Primers specific for SLCO2B1 and SERPINA1 were used for the determination of nuclear DNA (nDNA), and two primers (ND1, NADH dehydrogenase subunit 1, and ND5) were used for the detection of mtDNA. qRT-PCR was performed, and the mtDNA copy number was calculated according to the manufacturer's protocol.

Mitochondrial Imaging
HaCaT cells (6.0 × 10 4 cells/mL) were seeded onto a µClear fluorescence black plate (Greiner-Bio One, Tokyo, Japan) and cultured for 24 h. The cells were further cultured for 48 h with daily addition of 10 µM resveratrol or 100 ng/mL essential oil. HaCaT cells were then treated with 2.

Fluorescent Senescence-Associated β-Galactosidase Assay
Irradiated or non-irradiated HaCaT cells (6.0 × 10 4 cells/mL) were seeded onto a µClear fluorescence black plate and cultured for 24 h. The cells were further cultured for 48 h with daily addition of 10 µM resveratrol or 100 ng/mL essential oil. The fluorescent senescence-associated β-galactosidase (SA-β-Gal) assay was performed using a fluorescent substrate of β-galactosidase (C12FDG (5-dodecanoylaminofluorescein di-β-Dgalactopyranoside); Setareh Biotech, Eugene, OR, USA) as previously described [20,23]. The image of each well was acquired using an IN Cell Analyzer 2200 (GE Healthcare). Hoechst 33342 staining and fluorescent SA-β-Gal staining were used to define the nuclear and whole-cell regions, and the cell number and SA-β-Gal activity were evaluated by using Developer (GE Healthcare).

Statistical Analysis
All experiments were performed at least three times, and the corresponding data are shown. The results are presented as mean ± standard deviation. Statistical significance was determined using a two-sided Student's t-test. Statistical significance was defined as p < 0.05 (* p < 0.05; ** p < 0.01; *** p < 0.001).

Conclusions
In the present study, essential oils derived from C. laurifolius and C. monspeliensis were found to augment the expression of SIRT1 in keratinocytes, increase the number and size of mitochondria, and augment mitochondrial activity. In addition, these essential oils were found to attenuate ultraviolet-B-induced mitochondrial damage and cellular senescence in keratinocytes. These findings demonstrate that essential oils derived from C. laurifolius and C. monspeliensis may inhibit skin aging through mitochondrial regulation via SIRT1 activation.